The goal of this research program is to elucidate the physical mechanism of genetic control processes mediated by the interactions of proteins at specific sites on DNA. Components of the program include: (a) development of new techniques for the study of site- specific protein-DNA interactions, (b) theoretical work on the thermodynamic of coupled individual-site binding reactions in multi-site gene control systems, (c) experimental studies of functional energetics in specific gene control systems, (d) correlation of the energetic properties with structural features of the interacting molecules, (e) development and testing of physical-chemical models for gene control systems. We plan to extend our previous studies on the repressor-operator control system of bacteriophage lambda, which is a prototype for a large family of cooperative multi-site gene control systems. Using the Quantitative DNase Footprint Titration technique that we have recently developed, we will carry out a detailed study of the effects of temperature, pH, and ionic interactions on the site- specific energetics (including cooperativity effects) of cI and cro repressor binding to the DNA operator sites. The results will provide new insights into the detailed roles of proton binding, cation binding, and the various non-covalent forces responsible for the regulatory interactions. We will study the biological role of cooperative interactions between operator-bound repressors by in vivo and in vitro studies of mutant repressors that have altered cooperativity, using a physical-chemical model of gene control. We also plan further development of theory and techniques for understanding site-specific protein-DNA interactions.